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Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants

Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity... Volume 2 † Number 1 † March 2009 10.1093/biohorizons/hzp002 ......................................................................................................................................................................................................................................... Research article Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants Richard Wheeler* Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK. * Corresponding author. Tel: þ44 0114 222 4413. Email: R.Wheeler@sheffield.ac.uk Supervisors: Dr Robert L. Davies and Dr Richard Burchmore, Faculty of Biomedical and Life Sciences, Division of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, UK. ........................................................................................................................................................................................................................................ Pasteurella multocida is a Gram-negative bacterium responsible for diseases affecting a broad range of farm-reared animals. Although there is an association between capsular serotype and disease, the molecular basis of host specificity is poorly understood. Outer mem- brane proteins (OMPs) are at the interface of bacterium and host and are likely to play important roles in host specificity and disease. Two classes are of particular importance—adhesins that are adapted for colonization of specific host niches and iron-acquisition proteins that allow pathogens to acquire iron from host-specific iron complexes. A comparative analysis of the outer membrane (OM) proteome of eight P. multocida isolates associated with disease of avian, bovine, ovine and porcine species was performed to identify putative host-specificity determinants. Isolates were cultured in iron-replete media, and also in iron-limited conditions to mimic the iron- limited host environment, and induce expression of iron-regulated OMPs expressed in vivo. The OMP-rich sarcosyl-insoluble cell fraction was isolated and the OMPs were separated by SDS-PAGE and identified by matrix-assisted light desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The expressed proteome was compared with the in silico predicted proteome from the genome sequence of P. multocida strain Pm70, using PSORTb and Proteome Analyst subcellular localization software. In iron-rich conditions iso- lates were clustered into three groups based on high molecular weight (HMW) OMP similarity. Isolates responsible for invasive disease were clustered into a single group. Putative colonization OMPs were present in isolates recovered from different host species, but showed molecular weight heterogeneity. Such proteins are good candidates for further study as disease or host-specificity determinants, as variation between these proteins may be a consequence of adaptation to different host niches. HMW OMPs were identified as being involved in iron-uptake. However, isolates associated with different diseases and host species expressed different iron-uptake proteins, or regulated expression differently, suggesting adaptation to specific host niches. Key words: Pasteurella multocida, outer membrane protein, host specificity, iron acquisition. ........................................................................................................................................................................................................................................ in cattle, pigs and sheep in western countries, as well as Introduction cases of meningitis, abortion, localized infections and masti- 2–4 The bacterium Pasteurella multocida is a Gram-negative tis. Asymptomatic carriers of P. multocida act as a reser- facultative anaerobe commonly found as a commensal in voir of infection, and transmission is thought to occur by the upper respiratory tract of a diverse range of mammalian direct contact with infected animals, or dissemination in 1 5 and avian species. Many strains are opportunistic agents of water supplies. primary or secondary disease, resulting in economically sig- The association of particular strains of P. multocida with nificant outbreaks affecting farm-reared animals. Disease specific disease syndromes is complex. Strains can be syndromes include fowl cholera of poultry, progressive grouped serologically into 5 capsular types (A, B, D, E atrophic rhinitis (PAR) in pigs, haemorrhagic septicaemia and F) and 16 somatic lipopolysaccharide-types (1–16). of cattle in Asia and Africa, and pneumonic pasteurellosis Previous studies have reported an association between the ......................................................................................................................................................................................................................................... 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... capsular serogroup and disease, although certain serogroups host in vivo conditions and facilitate the identification of are associated with a range of diseases in different host iron-regulated OMPs. species. P. multocida strains have also been characterized by outer membrane protein (OMP)-type and 16S Materials and methods rRNA-type. 16S rRNA-typing revealed that the majority of clinical isolates belong to a single lineage containing Bacterial isolates and culture conditions seven 16S-types. However a range of capsular types, Eight P. multocida isolates commonly associated with invasive OMP-types and host species were represented, indicating sig- or pneumonic disease syndromes in avian, bovine, ovine or nificant heterogeneity between closely related strains. Disease porcine hosts, and representing a range of serotypes and progression is therefore thought to depend on a complex OMP-types, were included in this study (Table 1). Pure interaction of host factors including species, age and P. multocida cultures were obtained by plating 10 ml bacterial immune status, and strain-dependent virulence factors such suspensions from frozen glycerol stocks [50% (v/v) glycerol in as production of toxins, adhesins and mechanisms for brain-heart infusion broth (BHIB); Oxoid] onto blood agar acquiring nutrients from the host. plates [5% defibrinated sheep’s blood in brain-heart infusion OMPs are key mediators of bacterial interaction with the agar (BHIA); Oxoid] and incubating at 378C for 24 h. host environment. Until recently, characterized OMPs included the major proteins OmpA and OmpH and a Optimization of iron-limited culture conditions limited number of minor OMPs that were investigated as Isolates were grown in BHIB containing the iron-chelating virulence factors or immunogens. Completion of the agent 2,2 -dipyridyl to reduce the concentration of iron avail- genome sequence of an avian serogroup A:3 strain, able in the growth media. Dipyridyl concentrations capable P. multocida strain Pm70, in conjunction with the develop- of inducing observable expression of iron-uptake OMPs ment of in silico proteome prediction tools, has facilitated without completely inhibiting growth were determined by a bioinformatics-based approach to the study of OMPs. growth curves conducted in 100 ml BHIB containing 0, 50, OMP expression can now be considered within the context 100, 150 or 200 mM 2,2 -dipyridyl. Growth curves were of the full OM-proteome complement. Recent studies have repeated over a range of dipyridyl concentrations where taken advantage of this technology to investigate in vivo small changes in dipyridyl concentration produced a rapid and in vitro OMP expression via mass spectrometry (MS) decline in growth. The final concentrations of 2,2 -dipyridyl and microarrays, and to identify candidate immunogens for selected for batch culture of each isolate were PM144, 9, 11, 12 reverse vaccine development. However, OMP 100 mM; PM246, 140 mM; PM564, 120 mM; PM632, expression of isolates from different host species has not 100 mM; PM684, 100 mM; PM734, 60 mM; PM966, been investigated. 100 mM; PM982, 100 mM. This study aimed to characterize the OM proteome of P. multocida isolates from different host species and to ident- Sarcosyl extraction of OMPs ify OMPs that may contribute to host specificity. OMPs OMP fractions were prepared using the sarcosyl extraction present in isolates from different host species are likely to 16, 17 method described previously. Inocula were prepared have an essential function for bacterial survival. A compara- by inoculating five to six colonies into 15 ml BHIB and tive analysis of the OM proteome of eight P. multocida iso- lates recovered from avian, bovine, ovine and porcine hosts Table 1. Properties of Pasteurella multocida isolates was performed. OMPs were isolated by sarcosyl extraction Isolate Host Serotype OMP 16S Associated and OMP profiles compared by SDS-PAGE. OMPs of inter- a a a designation origin type type disease ................................................................................................................ est were identified and compared by MALDI-TOF-MS, a PM144 Avian A 1.1 2 Septicaemia rapid, high throughput method capable of measuring peptide mass to 0.01% accuracy. OMPs expressed by iso- PM246 Avian F 2.2 2 Septicaemia lates from different host species were compared against the PM564 Bovine A 2.1 3 Pneumonia predicted OM proteome of P. multocida strain Pm70 using PM632 Bovine A 4.1 1 Pneumonia Proteome Analyst (PA) and PSORTb software. PA predicts PM684 Porcine A 6.1 2 Suspected protein features such as subcellular localization from PAR SWISS-PROT gene annotation key words of sequence homo- PM734 Porcine A 1.1 2 Pneumonia logues, providing high coverage of Gram-negative pro- PM966 Ovine A 1.1 1 Pneumonia teomes, whereas PSORTb is a high precision tool PM982 Ovine D 3.1 1 Pneumonia combining multiple primary sequence features to predict 14, 15 protein features. Isolates were also cultured in iron- a 6 Data taken from a previous study by Davies. replete conditions and iron-limited conditions to replicate PAR, Porcine atrophic rhinitis. ......................................................................................................................................................................................................................................... 2 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... incubating overnight at 378C with shaking at 120 rpm. For 4% (w/v) SDS, 10% (v/v) 2-b-mercaptoethanol, 0.5% batch culture, 400 ml of pre-warmed BHIB were inoculated (w/v) bromophenol blue] and heated in a boiling water with 800 ml (1:500) of overnight culture and incubated at bath for 5 min. SDS-PAGE was carried out using a Protean 378C with shaking at 120 rpm, until cultures reached I Dual Slab apparatus (Bio-Rad, Richmond, CA94804). mid-log phase equivalent to an OD of 1.0 (6–8 h The gel comprised a 4% acrylamide stacking gel and a 600 nm of growth). For iron-limited growth conditions, the appropri- 12% acrylamide resolving gel. A Tris-glycine reservoir ate concentration of sterile 2,2 -dipyridyl was added. Growth buffer (25 mM Tris Base, 192 mM glycine and 0.1% SDS; curves were generated by removing 1 ml of culture every 1– pH 8.3) was used. Twenty micrograms of OMP samples 2 h and measuring OD with a WPA Biowave CO8000 were loaded into each lane and electrophoresis conducted 600 nm cell density meter. On achieving mid-log phase, cultures were using a constant current of 20 mA per gel through the stack- placed on ice water for 5 min to stop growth. Purity checks ing gel and 30 mA per gel through the resolving gel. Protein were performed by plating out one drop of culture onto bands were visualized by staining with Coomassie blue. Gels blood agar and incubating for 24 h at 378C. Cultures were were stored in 5% (v/v) acetic acid. harvested by centrifugation at 10 000g for 30 min at 48C. Preparation of proteins for MALDI-TOF-MS The bacterial pellet was resuspended in 50 ml ice cold 20 mM Tris-HCl (pH 7.2) and centrifugated at 10 000g Thirty-six protein bands expressed in iron-rich culture were for 30 min at 48C. The pellet was resuspended in 8.0 ml selected for identification and 38 bands were selected from iso- of ice-cold 20 mM Tris-HCl (pH 7.2) and the cells were lates grown in iron-limited media. Protein bands were excised lysed by sonication for 5 min in ice water using a Soniprep for in situ trypsin digestion. Gel pieces were washed in sonicator at 12 mm amplitude. Unbroken cells were 100 mM ammonium bicarbonate for 1 h and washed again removed by centrifugation at 48C for 30 min at 10 000g. with acetonitrile/100 mM ammonium bicarbonate (1:1). The supernatants were carefully transferred to 10 ml ultra- Cysteine reduction was achieved by incubating samples in centrifuge tubes and cell envelopes pelleted by centrifugation 160 ml45mM DTT/100 mM ammonium bicarbonate at 50 000g for 1 h at 48C. The gelatinous pellets were fully (1:15) at 608C for 30 min. Samples were cooled to room temp- resuspended in 10 ml 0.5% sodium N-lauroylsarcosine erature and alkylated by adding 10 ml of 100 mM iodoaceta- (sarcosyl) using long-form Pasteur pipettes, and the mide and incubating in darkness at room temperature for sarcosyl-insoluble OM fraction was pelleted by centrifu- 30 min. Gel slices were washed for 1 h in acetonitrile/ gation at 50 000g for 1 h at 48C. The OM pellet was 100 mM ammonium bicarbonate (1:1), dehydrated in aceto- resuspended in 10 ml 20 mM Tris-HCl (pH 7.2) and centri- nitrile for 10 min and dried in a Speedvac vacuum centrifuge. fugated again at 50 000g for 1 h at 48C. The final OM pellet Gel pieces were fully rehydrated at 48C in 25 mM ammonium was carefully resuspended in a small volume (less than 1 ml) bicarbonate containing 0.2 mg/ml sequence grade modi- of 20 mM Tris-HCl (pH 7.2) to achieve a final concentration fied porcine trypsin (Promega, V111). Sufficient 25 mM greater than 2.0 mg/ml and stored at –208C. ammonium bicarbonate was added to cover the gel pieces. Proteins were digested overnight at 378C. Trypsin digested pep- Modified Lowry assay for the determination of OMP tides were extracted by adding an equal volume of acetonitrile concentration to the digest, incubating at room temperature for 20 min and The modified assay improves solubilization of membrane pro- transferring the eluate to a 96-well plate. The extraction was teins. Fifty microlitre volumes of OM samples were made up repeated using 1% formic acid. Pooled peptide extracts were to 1 ml with distilled water in a test tube. Three millilitres of precipitated by vacuum centrifugation at 458C. The precipitate alkaline copper reagent (2.0% Na CO , 0.4% NaOH, was resuspended in 4 mlof 50% (v/v) acetonitrile, 0.1% (v/v) 2 3 0.16% sodium tartrate, 1.0% SDS, 0.04% CuSO .5H O) trifluoroacetic acid (TFA) and 1 ml mixed with an equal 4 2 were added and the tubes vortexed and incubated at room volume of matrix comprising a saturated solution of cyano-4- temperature for 60 min. Three hundred microlitres of 1:1 hydroxycinnamic acid (CHCA; Bruker Daltonics GmBH, dilute Folin-Ciocalteu phenol reagent were added and the Germany) in 50% (v/v) acetonitrile, 0.1% (v/v) TFA on a tubes vortexed and incubated for a further 45 min at room polished stainless steel target plate (MTP 384 target plate, temperature. Absorbance was measured at 660 nm on a Bruker Daltonics GmBH, Germany) and allowed to air dry Unicam UV/VIS spectrophotometer. A standard curve was at room temperature. generated from triplicate bovine serum albumin (BSA) Peptide mass fingerprinting by MALDI-TOF-MS protein standards in the range 0.0–1.0 mg/ml. OMP stocks were then adjusted to a final concentration of 2.0 mg/ml. OMPs were analysed using a Voyager DEpro MALDI- TOF-MS (Applied Biosystems). Mass spectra were internally SDS-PAGE of OMPs calibrated against autologous trypsin peptide peaks. Data OMP samples were adjusted to 1.0 mg/ml in 2 sample were collected over the mass range 800–4000 Da, using a buffer [0.125 M Tris-HCl (pH 6.8), 20% (v/v) glycerol, minimum signal-to-noise ratio filter of 10. OMPs were ......................................................................................................................................................................................................................................... 3 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... identified from the peptide mass fingerprinting (PMF) output from lane 5 (PM684) and lane 8 (PM982) were correctly by the MASCOT (Matrix Science) search engine. The data- identified as OmpA and OmpH by PMF. It was noted that base search was restricted to the phylum Proteobacteria and proteins in the HMW OMP region (85–100 kDa) formed allowed for a maximum of one missed cleavage, modification three groups, comprising either three weakly expressed by carbaridomethylation, variable modification of methion- OMPs (Fig. 1, lanes 1, 2 and 5), two weakly expressed ine residues by oxidation and a positive peptide charge of 1. OMPs and one lower MW, strongly expressed OMP (Fig. 1, lanes 3, 4 and 8) or five moderately expressed Prediction of OMPs from genome sequence OMPs (Fig. 1, lanes 6 and 7). When isolates were clustered OMPs identified by MS were compared with proteins pre- by HMW OMP homology, there was an association dicted to have an OM subcellular localization from the com- between HMW OMP-type and disease-type (Fig. 2). plete genome sequence of P. multocida strain Pm70 Isolates responsible for invasive diseases (avian PM144 and (GenBank accession no. AE004439). To maximize cover- PM246, fowl cholera; porcine PM684, PAR) formed a age of the OM proteome, OMP predictions were generated single distinct cluster. The remaining isolates formed two from the combined results of analysis of the Pm70 genome clusters associated with pneumonic pasteurellosis. using PA v.3.0 (http://www.cs.ualberta.ca/~bioinfo/PA/) MALDI-TOF-MS identified that OMPs in the HMW and PSORTb v.2.0 (http://www.psort.org/psortb) software. region were mainly involved in iron acquisition. The two All PSORTb OM subcellular localization predictions had a groups of isolates associated with pneumonic disease confidence value .9.0. expressed different iron-acquisition proteins depending on the HMW OMP-type. The group comprising both bovine isolates (PM564 and PM632) and ovine isolate PM982 Results expressed a haemoglobin-binding protein (HgbA) and SDS-PAGE and MALDI-TOF-MS of OMPs from iron-rich transferrin-binding protein (TbpA). The second pneumonic culture group, comprising porcine isolate PM734 and ovine isolate PM966, expressed haem-acquisition receptors HemR and OMP profiles comprised two major proteins (OmpA and PfhR. The OMP profiles of PM734 and PM966 were OmpH) and 11–14 minor proteins (Fig. 1). Positive controls Figure 1. Coomassie blue-stained SDS-PAGE gel showing OMP profiles of Pasteurella multocida isolates of avian, bovine, porcine and ovine origin. Isolates were grown in iron-rich media. Lane numbers are given at the top of the gel. Isolate designation and host of origin is stated at the bottom. Lane 9 shows the molecular weight (MW) standards (kDa). Labelled bands correspond to OMPs of interest discussed in the main text and were identified by MALDI-TOF-MS. ......................................................................................................................................................................................................................................... 4 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... Figure 2. SDS-PAGE profiles of Pasteurella multocida isolates grown in iron-rich media, clustered by homology of HMW OMPs. Isolates associated with invasive diseases clustered into a single group. Pneumonic isolates formed two distinct clusters. Disease type is indicated at the top of the gel. Isolate designation and host of origin is stated at the bottom. MW standards (kDa) are indicated. The HMW OMP region is highlighted, as are proteins PM1069 and OMP16 that were present in all isolates but demonstrated MW variation. generally very similar, and both also expressed a protein with iron-transport OMP (Fig. 3). HemR and PfhR were common a moderate degree of homology to a Shewanella pealeana to all invasive disease isolates. Porcine isolate PM684 expressed type IV pilus biogenesis protein PilZ (Fig. 1). Only one an additional OMP, HasR, which was not identified from the HMW OMP of the invasive disease cluster was successfully invasive avian isolates, but was expressed by the other porcine identified by MS at this point and was an organic solvent tol- isolate PM734, which was associated with pneumonic disease. erance (Ost) protein. HMW OMP expression by porcine isolate PM734 increased in response to iron limitation, and six bands were SDS-PAGE and MALDI-TOF-MS of OMPs from iron-limited visible compared with five bands in iron-rich culture. culture Expressed iron-uptake OMPs identified were HasR, HemR, PfhR and PM0741 (Fig. 4, Tables 2 and 3). Ovine isolate Although invasive disease isolates PM144, PM246 and PM966, which had a very similar OMP profile to PM734 PM684 and pneumonic isolates PM734 and PM966 in iron-rich culture, had reduced HMW OMP expression formed distinct groups by HMW OMP similarity following (four bands visible), although expression of HemR, PfhR iron-rich culture, expression of HMW OMPs by these and PM0741 was confirmed (Fig. 4, Tables 2 and 3). groups was altered in response to iron-limitation (Fig. 3). Isolates that were previously similar by HMW OMP-type OMPs demonstrating MW variation had different HMW OMP profiles depending on the host of origin (Fig. 4, Tables 2 and 3). In contrast, OMP MS of proteins expressed following iron-rich or iron-limited expression by the pneumonic disease cluster comprising culture identified four proteins that were present in isolates bovine isolates PM564, PM632 and ovine isolate PM982 from different host species but in variant MW forms. A con- was unaffected by iron limitation. In particular, there was served band of 47.3 kDa was identified as hypothetical no significant change in expression of iron-acquisition pro- protein PM1069 (Fig. 1). MW variation of PM1069 was teins TbpA or HgbA. observed for isolates from different hosts and was most MS of proteins in the HMW region revealed that several notable for ovine isolate PM982 which possessed a iron-acquisition OMPs were expressed by isolates from the 44.2 kDa MW variant (Fig. 2). A 17.9 kDa conserved invasive disease group and the PM734/PM966 pneumonic protein was identified as OMP16 (Fig. 1). PM564 expressed group. These included haem-acquisition OMPs HasR, an 18.1 kDa form, and PM246 a 19.2 kDa form (Fig. 2). A HemR, PfhR and hypothetical protein PM0741, a putative 40.7 kDa OMP, HexD, was conserved across species but was ......................................................................................................................................................................................................................................... 5 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 3. Coomassie blue-stained SDS-PAGE showing OMP profiles of Pasteurella multocida isolates of avian, bovine, porcine and ovine origin, following growth in iron-limited conditions. Lane numbers are given at the top of the gel. Isolate designation and host of origin is stated at the bottom. Lane 9 shows the MW standards (kDa). Labelled bands denote OMPs of interest discussed in the main text and were identified by MALDI-TOF-MS. only weakly expressed by avian isolates and porcine isolate inorganic ion and metabolic trafficking (12 OMPs), and PM684 (Fig. 3). Expression of PM1069, OMP16 and secretion and intracellular transport (8 OMPs). A quarter HexD was not affected by iron limitation. MS of HMW of predicted OMPs were of unknown function. OMPs revealed that OMP87, which is not associated with Comparison of expressed and predicted P. multocida OM iron-uptake, was conserved across isolates from all four proteome hosts (Fig. 3). However, expression levels were difficult to compare due to the tight clustering of bands and altered In total, 22 different OMPs were shown to be expressed by expression of proteins in the HMW region under iron-rich P. multocida isolates from different hosts under iron-rich or or iron-limited conditions. iron-limited growth conditions. A small number of proteins identified by MS were not identified by the proteome predic- The in silico predicted OM proteome tion software, including the major protein OmpA and Analysis of the P. multocida strain Pm70 genome sequence OMP16. This may have been due to differences in nomencla- by PSORTb and PA software identified a total of 79 proteins ture. Prediction software identified multiple occurrences of predicted to have an OM localization. Thirty-nine OMPs OmpH (three occurrences), Hsf (two occurrences) and LspB were identified by both prediction algorithms, whereas 11 (two occurrences). Expression of all definitively identified OMPs were predicted only by PSORTb and 29 by PA iron-uptake OMPs predicted from the Pm70 genome was con- alone. Predicted OMPs represented 12 functional classes by firmed in different isolates. However, whereas only three clusters of orthologous groups (COGs) (Fig. 5). The major definitive iron-acquisition OMPs were predicted from the groups identified were cell envelope biogenesis (31 OMPs), Pm70 genome (HasR, HemR and PfhR), MS identified five ......................................................................................................................................................................................................................................... 6 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... Figure 4. OMP profiles of Pasteurella multocida isolates cultured in iron-rich or iron-limited media resolved by Coomassie blue-stained SDS-PAGE. Isolates are grouped by HMW OMP similarity under iron-rich culture conditions. Isolate ID, host species, disease type, iron availability and MW standards (kDa) are indicated. Numbered bands denote OMPs identified by MALDI-TOF-MS. Corresponding band identities are shown in Tables 2 (iron-rich culture) and 3 (iron-limited culture). iron-uptake OMPs (HasR, HemR, PfhR, HgbA and TbpA). Eight hypothetical proteins were predicted to have HgbA and TbpA were not predicted from the avian Pm70 iron-uptake functions. Expression of hypothetical OMPs genome, but were expressed by bovine isolates PM564 and PM0300, PM0336 and PM0741 (putative iron-uptake PM632 and ovine isolate PM966. OMPs by COGs classification) was confirmed (Fig. 4, ......................................................................................................................................................................................................................................... 7 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Table 2. Identification of selected Pasteurella multocida OMPs from iron-rich culture, determined by Mascot and BLASTP analysis Band Accession No. Protein name Molecular Protein Matched Coverage identity weight score peptides (%) ....................................................................................................................................................................................................................................... PM144.1 15214145 47kDa OMP precursor 46 032.7 219 19 56 15602934 PM1069 47 831.6 217 19 54 PM144.2 49182350 OmpA 37 785.4 85 17 61 PM246.1 15602651 PM0786 38 121.6 129 25 73 49182354 OmpA 38 125.6 102 22 67 PM564.1 15602165 PM0300 110 044.6 153 42 47 33340606 HgbA 110 664.9 125 37 44 PM564.2 13027667 TbpA 89 377.0 547 33 47 PM564.3 6977946 OMP 16 protein 14 207.0 180 6 73 PM632.1 15214145 47 kDa OMP precursor 46 032.7 108 18 60 15602934 PM1069 47 831.6 106 18 58 PM632.2 6977946 OMP 16 protein 14 207.0 140 8 82 PM684.1 25008882 LPS-assembly precursor (Ost) 90 819.4 88 26 40 PM684.2 15602651 PM0786 38 121.6 114 20 66 49182354 OmpA 38 125.6 85 16 54 PM684.3 2853254 OmpH 34 656.9 105 18 54 PM734.1 15602441 HemR 85085.8 330 26 39 PM734.2 157962851 PilZ [S. pealeana ATCC 92 441.9 78 28 41 700345] PM966.1 7716525 Putative TonB-dependent 110 287.5 79 33 35 receptor PM966.2 15602441 HemR 85 085.8 187 22 33 15601905 PfhR 81 510.1 182 24 30 PM966.3 15214145 47 kDa OMP precursor 46 032.7 84 20 60 15602934 PM1069 47 831.6 82 20 58 PM966.4 157962851 PilZ [S. pealeana ATCC 92 441.9 76 27 40 700345] PM982.1 110725170 TbpA 89 399.2 329 36 53 PM982.2 15214145 47 kDa OMP precursor 46 032.7 78 18 57 15602934 PM1069 47 831.6 76 18 55 PM982.3 99083546 OMP 38 821.3 123 15 40 2853254 OmpH 34 656.9 83 13 39 Band identities correspond to Fig. 4. Bands in bold are the nearest characterized protein identification when the highest scoring OMP was a poorly characterized or hypothetical protein (HP). Tables 2 and 3). A further five hypothetical proteins with between animal species. However, the role of specific adhe- probable iron receptor functions were predicted from the sins in host colonization and disease has not been well 21, 22 Pm70 genome (PM0337, PM0745, PM1081, PM1282, characterized. Study of a bovine P. multocida strain PM1428), but expression was not confirmed. implicated fibronectin (Fn) as a specific receptor for P. multocida and identified five OMPs involved in binding to host Fn. Fn is a dimer that occurs in different isoforms and has eight functional domains that may be exploited as Discussion receptors for bacterial colonization. Two classes of Fn have Candidate adhesins of P. multocida been identified—a soluble form present in body fluids and 23 20 an ECM form. Dabo et al. showed that bovine strains It has been proposed that the broad host range of preferentially bound the N-terminal Hep 1 domain. In the P. multocida is due to colonization by adherence to extra- current study, three of the OMPs identified previously, cellular matrix (ECM) components that are ubiquitous ......................................................................................................................................................................................................................................... 8 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... Table 3. Identification of selected Pasteurella multocida OMPs from iron-limited culture, determined by Mascot and BLASTP analysis Band ID Accession No. Protein name Molecular weight Protein score Matched peptides Coverage (%) ........................................................................................................................................................................................................................................ PM144.1 145632020 CGSHi22421_00657 90 430 90 13 21 [H. influenzae] PM144.2 15603857 HP PM1992 87 822 250 28 45 27527748 Omp87 87688 222 26 41 PM144.3 15601905 PfhR 81 510 179 24 39 PM144.4 15603595 PlpB 30 276 110 12 59 PM144.5 15602863 HP PM0998 30 915 89 9 44 PM246.1 15602201 HP PM0336 114 285 224 29 35 PM246.2 15603487 HasR 96 079 331 35 50 PM246.3 15603857 HP PM1992 87 822 132 20 32 27527748 Omp87 87 688 120 19 29 PM246.4 15601905 PfhR 81 510 264 29 51 PM246.5 15214145 47 kDa OMP precursor 46 033 212 21 69 15602934 HP PM1069 47 832 209 21 67 PM246.6 15602651 HP PM0786 38 122 172 21 51 49182354 OmpA 38 126 147 18 45 PM246.7 15603408 HP PM1543 26 864 97 11 60 PM246.8 6977946 OMP 16 protein 14 207 115 8 90 PM564.1 13027667 TbpA 89 377 254 28 45 PM632.1 33340606 HgbA 110 665 148 22 29 PM632.2 15603857 HP PM1992 87 822 307 33 53 27527748 Omp87 87 688 293 32 53 PM632.3 13027667 TbpA 89 377 350 36 54 PM632.4 15602643 HexD 43 034 123 14 52 PM632.5 15602863 HP PM0998 30 915 86 9 41 15214146 24 kDa OMP precursor 28 719 78 8 35 PM684.1 15603487 HasR 96 079 369 37 51 PM684.2 15602668 HP PM0803 91 050 147 22 32 PM684.3 15601905 PfhR 81 510 271 30 55 PM734.1 15603487 HasR 96 079 248 29 47 PM734.2 27527748 Omp87 87 688 119 18 32 PM734.3 15601905 PfhR 81 510 268 30 50 PM734.4 15602643 HexD 43 034 131 15 60 PM966.1 15603857 HP PM1992 87 822 302 32 50 27527748 Omp87 87 688 288 31 51 PM966.2 15602606 HP PM0741 89 716 114 19 30 PM966.3 15601905 PfhR 81 510 182 24 40 PM966.4 15602863 HP PM0998 30 915 107 9 40 15214146 24 kDa OMP precursor 28 719 98 8 34 PM982.1 110725170 TbpA 89 399 250 30 48 Band identities correspond to Fig. 4. Bands in bold are the nearest characterized protein identification when the highest scoring OMP was a poorly characterized or hypothetical protein (HP). PM1069 (P80603), OMP87 and OMP16, were conserved present across different isolates but displayed MW hetero- between isolates from different host species, providing evi- geneity. OMPs that display MW heterogeneity across isolates dence of the importance of these OMPs to the broad host from different hosts are of particular interest, as the observed range of P. multocida. OMPs PM1069 and OMP16 were variation may have arisen due to selective pressures acting on ......................................................................................................................................................................................................................................... 9 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 5. COGs classification of OMPs predicted from the OM proteome of Pasteurella multocida strain Pm70. A total of 79 OMPs were predicted using PSORTb and PA software. these OMPs within different host niches. These proteins are disease. Other authors have previously speculated that therefore useful candidates for further study as biofilm formation by P. multocida may be important in host-specificity determinants. For example, MW hetero- establishing disease, but this area has not been well geneity between these OMPs could reflect recognition of studied. Fn-types encountered in different host niches during invasive HMW OMP profile and host specificity or pneumonic disease, variation in the abundance of Fn iso- forms found within particular host species, or recognition of When isolates were cultured in iron-rich media, they clus- different Fn domains. tered into three groups by HMW OMP homology and Pilus structures expressed by P. multocida have been there was an association between HMW OMP-type and implicated as the primary mechanism for colonization of disease, as all invasive disease isolates clustered into a nasal epithelia, but there are few studies to support this single group. It is possible that homology of HMW OMPs hypothesis. A protein with homology to the type IV pilus expressed in a nutrient-rich environment reflects an epide- assembly protein PilZ of S. pealeana was present in the miological relationship between these isolates. Nutrient- OMP profile of PM734 and PM966. However, other poten- rich culture may elicit a basal state of OMP expression, as tial pilus components, such as those comprising the OM inte- there is no pressure for increased expression of a particular gral O-ring, were not identified. In addition, putative class of uptake system. This would explain why isolates filamentous adhesin Hsf and haemagglutinins PfhB1 and from different host species had similar HMW OMP profiles PfhB2 were predicted from the Pm70 genome and may be in a nutrient-rich environment, but had dissimilar HMW important virulence determinants, but were not detected in OMP profiles under iron-limitation stress. the present study. This could have been due to incomplete The results also suggested that expression or regulation of identification of the OM proteome, lack of expression in iron-uptake OMPs may be adapted for survival within par- vitro or because the vigorous methods involved in sarcosyl ticular host species or specific host niches. Avian isolates extraction are inappropriate for the isolation of fragile fila- PM144 and PM246 were both associated with septicaemia mentous surface structures. and had very similar OMP profiles under iron-limited con- Expression of type IV pili by P. multocida has been ditions. However, porcine isolates PM684 and PM734 characterized for serogroups A, B and D, and is responsible were associated with diseases (PAR and pneumonia respect- for twitching motility. Twitching motility is an important ively) that occur at different sites within the host, and had step in biofilm formation, for example during lung infections different HMW OMP profiles under iron limitation. by Pseudomonas aeruginosa in cystic fibrosis patients. It is Haem is the most abundant iron source in birds and feasible that biofilm formation is also important during lung mammals. Avian isolates PM144, PM246, porcine isolates infections by P. multocida, which would link expression of PM684, PM734 and ovine isolate PM966 expressed an array type IV pili to the ability of isolates to cause pneumonic of OMPs involved in the acquisition of iron via haem-uptake ......................................................................................................................................................................................................................................... 10 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... (HasR, HemR and PfhR). HasR is a haemophore receptor. proteome was expressed. While the expressed OM proteome Haemophore systems typically involve type I secretion of is expected to differ from the complete OM proteome due to the haemophore HasA into the extracellular environment, environmental and temporal regulation of expression, it has binding of host haem and delivery to the transmembrane been suggested that the majority of OMPs could fail detec- channel HasR. However, less efficient haem-uptake via tion due to low levels of expression. OMPs may also be direct binding of haem by HasR may also occur. BLAST mistakenly predicted from genes with unusual localization analysis revealed that the Pm70 genome does not contain signal sequences or from pseudogenes. In this study, three the hasA gene suggesting that at least some P. multocida iso- OmpH proteins (OmpH_1, OmpH_2, OmpH_3) were pre- lates utilize a HasA-independent mechanism. HemR and dicted. However, comparison against the OmpH_1 amino PfhR are thought to be haem-transport porins which bind acid sequence by BLAST analysis revealed that OmpH_2 different haem-containing compounds directly. The presence and OmpH_3 contain sequence gaps and are potentially of multiple haem-uptake mechanisms may broaden the range non-functional pseudogenes. of haem sources accessible by these isolates. In conclusion, 22 OMPs were identified from the OM pro- Haem-uptake systems are often associated with haemoly- teome of eight P. multocida isolates recovered from different sins that liberate haem from host erythrocytes and other cell host species. Expression of several hypothetical OMPs pre- types. P. multocida is described as non-haemolytic under dicted from the P. multocida Pm70 proteome was confirmed aerobic conditions. However, recent studies have reported by PMF. A variety of potential host colonization factors were production of an inner membrane-bound haemolysin identified, some of which were present in isolates from differ- AhpA, expressed under anaerobic conditions in vitro ent host species and occurred as MW variants (e.g. OMP16, 30, 31 and is most active against avian red blood cells. PM1069). HMW OMPs were mainly involved in iron Furthermore, a haemolysin secretion and activation protein acquisition. In iron-replete conditions, isolates clustered LspB was predicted from the Pm70 genome by both into three groups by HMW OMP profile, and there was an PSORTb and PA. The role of the putative AhpA virulence association between these groups and disease. However factor in host pathogenesis is unknown, but it is conceivable within these clusters, isolates from different host species that haemolysin production could enhance haem acquisition regulated iron-acquisition OMPs differently in response to under certain in vivo conditions and could be associated with iron limitation, suggesting that similar isolates may have increased virulence of strains. evolved alternate mechanisms for regulating expression of Bovine isolates PM564, PM632 and ovine isolate PM982 iron-acquisition OMPs. This may reflect adaptation for sur- expressed a unique set of iron-acquisition OMPs, HgbA and vival within different host niches. TbpA, which were not expressed by avian or porcine isolates and were not predicted from the avian Pm70 genome. Acknowledgements Acquisition of iron from host transferrin (Tf) typically involves two components, a lipoprotein TbpB, which is a I wish to thank the staff at the Glasgow Biomedical Research high-specificity receptor for host Tf, and a 100 kDa tonB- Centre and the Sir Henry Wellcome Functional Genomics dependent iron transport channel TbpA. TbpB-independent Facility for their support during this project, with special Tf binding can occur, but is less efficient. The present thanks to Dr. Robert L. Davies, Dr. Richard Burchmore study concurs with previous reports that P. multocida and Jay Jayawarzdena. expresses an 82 kDa form of TbpA and does not express TbpB at all, and furthermore, that TbpA is expressed only by bovine and ovine isolates. In this study, TbpA was con- Funding stitutively expressed in iron-rich and iron-limited conditions. Funding for this project was provided by the Faculty of This may be because the P. multocida tbp locus lacks the Biomedical and Life Sciences, University of Glasgow. Fur-regulated promoter which is usually upstream of tbpB and is therefore not regulated by iron availability. The presence of a neighbouring insertion sequence suggests that References the tbpA gene may have been acquired by horizontal 1. Rimler RB, Rhoades KR (1989) Pasteurella multocida.In CF Adlam, JM Rutter, transmission. eds, Pasteurella and Pasteurellosis. London: Academic Press, pp. 37–73. 2. Chanter N, Rutter JM (1989) Pasteurellosis in pigs and the determinants of Comparison of in vitro and in silico OM proteomics virulence of toxigenic Pasteurella multocida. In CF Adlam, JM Rutter, eds, The OMPs identified in this study represented around one- Pasteurella and Pasteurellosis. London: Academic Press, pp. 161–195. quarter of the OMPs predicted from the Pm70 genome. 3. Frank GH (1989) Pasteurellosis of cattle. In CF Adlam, JM Rutter, eds, This proportion may have been higher if all protein bands Pasteurella and Pasteurellosis. London: Academic Press, pp. 197–222. had been subjected to MS. A similar study by Boyce et al. 4. Rhoades KR, Rimler RB (1989) Fowl cholera. In CF Adlam, JM Rutter, eds, suggested that around one-third of the predicted OM Pasteurella and Pasteurellosis. London: Academic Press, pp. 95–113. ......................................................................................................................................................................................................................................... 11 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... 5. 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Anal Biochem 87: 206–210. J Proteome Res 2: 303–311. ........................................................................................................................................................................................................................................ Submitted on 30 September 2008; accepted on 19 January 2009; advance access publication 17 February 2009 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants

Wheeler, Richard
Bioscience Horizons , Volume 2 (1) – Mar 17, 2009
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Volume 2 † Number 1 † March 2009 10.1093/biohorizons/hzp002 ......................................................................................................................................................................................................................................... Research article Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants Richard Wheeler* Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK. * Corresponding author. Tel: þ44 0114 222 4413. Email: R.Wheeler@sheffield.ac.uk Supervisors: Dr Robert L. Davies and Dr Richard Burchmore, Faculty of Biomedical and Life Sciences, Division of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, UK. ........................................................................................................................................................................................................................................ Pasteurella multocida is a Gram-negative bacterium responsible for diseases affecting a broad range of farm-reared animals. Although there is an association between capsular serotype and disease, the molecular basis of host specificity is poorly understood. Outer mem- brane proteins (OMPs) are at the interface of bacterium and host and are likely to play important roles in host specificity and disease. Two classes are of particular importance—adhesins that are adapted for colonization of specific host niches and iron-acquisition proteins that allow pathogens to acquire iron from host-specific iron complexes. A comparative analysis of the outer membrane (OM) proteome of eight P. multocida isolates associated with disease of avian, bovine, ovine and porcine species was performed to identify putative host-specificity determinants. Isolates were cultured in iron-replete media, and also in iron-limited conditions to mimic the iron- limited host environment, and induce expression of iron-regulated OMPs expressed in vivo. The OMP-rich sarcosyl-insoluble cell fraction was isolated and the OMPs were separated by SDS-PAGE and identified by matrix-assisted light desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The expressed proteome was compared with the in silico predicted proteome from the genome sequence of P. multocida strain Pm70, using PSORTb and Proteome Analyst subcellular localization software. In iron-rich conditions iso- lates were clustered into three groups based on high molecular weight (HMW) OMP similarity. Isolates responsible for invasive disease were clustered into a single group. Putative colonization OMPs were present in isolates recovered from different host species, but showed molecular weight heterogeneity. Such proteins are good candidates for further study as disease or host-specificity determinants, as variation between these proteins may be a consequence of adaptation to different host niches. HMW OMPs were identified as being involved in iron-uptake. However, isolates associated with different diseases and host species expressed different iron-uptake proteins, or regulated expression differently, suggesting adaptation to specific host niches. Key words: Pasteurella multocida, outer membrane protein, host specificity, iron acquisition. ........................................................................................................................................................................................................................................ in cattle, pigs and sheep in western countries, as well as Introduction cases of meningitis, abortion, localized infections and masti- 2–4 The bacterium Pasteurella multocida is a Gram-negative tis. Asymptomatic carriers of P. multocida act as a reser- facultative anaerobe commonly found as a commensal in voir of infection, and transmission is thought to occur by the upper respiratory tract of a diverse range of mammalian direct contact with infected animals, or dissemination in 1 5 and avian species. Many strains are opportunistic agents of water supplies. primary or secondary disease, resulting in economically sig- The association of particular strains of P. multocida with nificant outbreaks affecting farm-reared animals. Disease specific disease syndromes is complex. Strains can be syndromes include fowl cholera of poultry, progressive grouped serologically into 5 capsular types (A, B, D, E atrophic rhinitis (PAR) in pigs, haemorrhagic septicaemia and F) and 16 somatic lipopolysaccharide-types (1–16). of cattle in Asia and Africa, and pneumonic pasteurellosis Previous studies have reported an association between the ......................................................................................................................................................................................................................................... 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... capsular serogroup and disease, although certain serogroups host in vivo conditions and facilitate the identification of are associated with a range of diseases in different host iron-regulated OMPs. species. P. multocida strains have also been characterized by outer membrane protein (OMP)-type and 16S Materials and methods rRNA-type. 16S rRNA-typing revealed that the majority of clinical isolates belong to a single lineage containing Bacterial isolates and culture conditions seven 16S-types. However a range of capsular types, Eight P. multocida isolates commonly associated with invasive OMP-types and host species were represented, indicating sig- or pneumonic disease syndromes in avian, bovine, ovine or nificant heterogeneity between closely related strains. Disease porcine hosts, and representing a range of serotypes and progression is therefore thought to depend on a complex OMP-types, were included in this study (Table 1). Pure interaction of host factors including species, age and P. multocida cultures were obtained by plating 10 ml bacterial immune status, and strain-dependent virulence factors such suspensions from frozen glycerol stocks [50% (v/v) glycerol in as production of toxins, adhesins and mechanisms for brain-heart infusion broth (BHIB); Oxoid] onto blood agar acquiring nutrients from the host. plates [5% defibrinated sheep’s blood in brain-heart infusion OMPs are key mediators of bacterial interaction with the agar (BHIA); Oxoid] and incubating at 378C for 24 h. host environment. Until recently, characterized OMPs included the major proteins OmpA and OmpH and a Optimization of iron-limited culture conditions limited number of minor OMPs that were investigated as Isolates were grown in BHIB containing the iron-chelating virulence factors or immunogens. Completion of the agent 2,2 -dipyridyl to reduce the concentration of iron avail- genome sequence of an avian serogroup A:3 strain, able in the growth media. Dipyridyl concentrations capable P. multocida strain Pm70, in conjunction with the develop- of inducing observable expression of iron-uptake OMPs ment of in silico proteome prediction tools, has facilitated without completely inhibiting growth were determined by a bioinformatics-based approach to the study of OMPs. growth curves conducted in 100 ml BHIB containing 0, 50, OMP expression can now be considered within the context 100, 150 or 200 mM 2,2 -dipyridyl. Growth curves were of the full OM-proteome complement. Recent studies have repeated over a range of dipyridyl concentrations where taken advantage of this technology to investigate in vivo small changes in dipyridyl concentration produced a rapid and in vitro OMP expression via mass spectrometry (MS) decline in growth. The final concentrations of 2,2 -dipyridyl and microarrays, and to identify candidate immunogens for selected for batch culture of each isolate were PM144, 9, 11, 12 reverse vaccine development. However, OMP 100 mM; PM246, 140 mM; PM564, 120 mM; PM632, expression of isolates from different host species has not 100 mM; PM684, 100 mM; PM734, 60 mM; PM966, been investigated. 100 mM; PM982, 100 mM. This study aimed to characterize the OM proteome of P. multocida isolates from different host species and to ident- Sarcosyl extraction of OMPs ify OMPs that may contribute to host specificity. OMPs OMP fractions were prepared using the sarcosyl extraction present in isolates from different host species are likely to 16, 17 method described previously. Inocula were prepared have an essential function for bacterial survival. A compara- by inoculating five to six colonies into 15 ml BHIB and tive analysis of the OM proteome of eight P. multocida iso- lates recovered from avian, bovine, ovine and porcine hosts Table 1. Properties of Pasteurella multocida isolates was performed. OMPs were isolated by sarcosyl extraction Isolate Host Serotype OMP 16S Associated and OMP profiles compared by SDS-PAGE. OMPs of inter- a a a designation origin type type disease ................................................................................................................ est were identified and compared by MALDI-TOF-MS, a PM144 Avian A 1.1 2 Septicaemia rapid, high throughput method capable of measuring peptide mass to 0.01% accuracy. OMPs expressed by iso- PM246 Avian F 2.2 2 Septicaemia lates from different host species were compared against the PM564 Bovine A 2.1 3 Pneumonia predicted OM proteome of P. multocida strain Pm70 using PM632 Bovine A 4.1 1 Pneumonia Proteome Analyst (PA) and PSORTb software. PA predicts PM684 Porcine A 6.1 2 Suspected protein features such as subcellular localization from PAR SWISS-PROT gene annotation key words of sequence homo- PM734 Porcine A 1.1 2 Pneumonia logues, providing high coverage of Gram-negative pro- PM966 Ovine A 1.1 1 Pneumonia teomes, whereas PSORTb is a high precision tool PM982 Ovine D 3.1 1 Pneumonia combining multiple primary sequence features to predict 14, 15 protein features. Isolates were also cultured in iron- a 6 Data taken from a previous study by Davies. replete conditions and iron-limited conditions to replicate PAR, Porcine atrophic rhinitis. ......................................................................................................................................................................................................................................... 2 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... incubating overnight at 378C with shaking at 120 rpm. For 4% (w/v) SDS, 10% (v/v) 2-b-mercaptoethanol, 0.5% batch culture, 400 ml of pre-warmed BHIB were inoculated (w/v) bromophenol blue] and heated in a boiling water with 800 ml (1:500) of overnight culture and incubated at bath for 5 min. SDS-PAGE was carried out using a Protean 378C with shaking at 120 rpm, until cultures reached I Dual Slab apparatus (Bio-Rad, Richmond, CA94804). mid-log phase equivalent to an OD of 1.0 (6–8 h The gel comprised a 4% acrylamide stacking gel and a 600 nm of growth). For iron-limited growth conditions, the appropri- 12% acrylamide resolving gel. A Tris-glycine reservoir ate concentration of sterile 2,2 -dipyridyl was added. Growth buffer (25 mM Tris Base, 192 mM glycine and 0.1% SDS; curves were generated by removing 1 ml of culture every 1– pH 8.3) was used. Twenty micrograms of OMP samples 2 h and measuring OD with a WPA Biowave CO8000 were loaded into each lane and electrophoresis conducted 600 nm cell density meter. On achieving mid-log phase, cultures were using a constant current of 20 mA per gel through the stack- placed on ice water for 5 min to stop growth. Purity checks ing gel and 30 mA per gel through the resolving gel. Protein were performed by plating out one drop of culture onto bands were visualized by staining with Coomassie blue. Gels blood agar and incubating for 24 h at 378C. Cultures were were stored in 5% (v/v) acetic acid. harvested by centrifugation at 10 000g for 30 min at 48C. Preparation of proteins for MALDI-TOF-MS The bacterial pellet was resuspended in 50 ml ice cold 20 mM Tris-HCl (pH 7.2) and centrifugated at 10 000g Thirty-six protein bands expressed in iron-rich culture were for 30 min at 48C. The pellet was resuspended in 8.0 ml selected for identification and 38 bands were selected from iso- of ice-cold 20 mM Tris-HCl (pH 7.2) and the cells were lates grown in iron-limited media. Protein bands were excised lysed by sonication for 5 min in ice water using a Soniprep for in situ trypsin digestion. Gel pieces were washed in sonicator at 12 mm amplitude. Unbroken cells were 100 mM ammonium bicarbonate for 1 h and washed again removed by centrifugation at 48C for 30 min at 10 000g. with acetonitrile/100 mM ammonium bicarbonate (1:1). The supernatants were carefully transferred to 10 ml ultra- Cysteine reduction was achieved by incubating samples in centrifuge tubes and cell envelopes pelleted by centrifugation 160 ml45mM DTT/100 mM ammonium bicarbonate at 50 000g for 1 h at 48C. The gelatinous pellets were fully (1:15) at 608C for 30 min. Samples were cooled to room temp- resuspended in 10 ml 0.5% sodium N-lauroylsarcosine erature and alkylated by adding 10 ml of 100 mM iodoaceta- (sarcosyl) using long-form Pasteur pipettes, and the mide and incubating in darkness at room temperature for sarcosyl-insoluble OM fraction was pelleted by centrifu- 30 min. Gel slices were washed for 1 h in acetonitrile/ gation at 50 000g for 1 h at 48C. The OM pellet was 100 mM ammonium bicarbonate (1:1), dehydrated in aceto- resuspended in 10 ml 20 mM Tris-HCl (pH 7.2) and centri- nitrile for 10 min and dried in a Speedvac vacuum centrifuge. fugated again at 50 000g for 1 h at 48C. The final OM pellet Gel pieces were fully rehydrated at 48C in 25 mM ammonium was carefully resuspended in a small volume (less than 1 ml) bicarbonate containing 0.2 mg/ml sequence grade modi- of 20 mM Tris-HCl (pH 7.2) to achieve a final concentration fied porcine trypsin (Promega, V111). Sufficient 25 mM greater than 2.0 mg/ml and stored at –208C. ammonium bicarbonate was added to cover the gel pieces. Proteins were digested overnight at 378C. Trypsin digested pep- Modified Lowry assay for the determination of OMP tides were extracted by adding an equal volume of acetonitrile concentration to the digest, incubating at room temperature for 20 min and The modified assay improves solubilization of membrane pro- transferring the eluate to a 96-well plate. The extraction was teins. Fifty microlitre volumes of OM samples were made up repeated using 1% formic acid. Pooled peptide extracts were to 1 ml with distilled water in a test tube. Three millilitres of precipitated by vacuum centrifugation at 458C. The precipitate alkaline copper reagent (2.0% Na CO , 0.4% NaOH, was resuspended in 4 mlof 50% (v/v) acetonitrile, 0.1% (v/v) 2 3 0.16% sodium tartrate, 1.0% SDS, 0.04% CuSO .5H O) trifluoroacetic acid (TFA) and 1 ml mixed with an equal 4 2 were added and the tubes vortexed and incubated at room volume of matrix comprising a saturated solution of cyano-4- temperature for 60 min. Three hundred microlitres of 1:1 hydroxycinnamic acid (CHCA; Bruker Daltonics GmBH, dilute Folin-Ciocalteu phenol reagent were added and the Germany) in 50% (v/v) acetonitrile, 0.1% (v/v) TFA on a tubes vortexed and incubated for a further 45 min at room polished stainless steel target plate (MTP 384 target plate, temperature. Absorbance was measured at 660 nm on a Bruker Daltonics GmBH, Germany) and allowed to air dry Unicam UV/VIS spectrophotometer. A standard curve was at room temperature. generated from triplicate bovine serum albumin (BSA) Peptide mass fingerprinting by MALDI-TOF-MS protein standards in the range 0.0–1.0 mg/ml. OMP stocks were then adjusted to a final concentration of 2.0 mg/ml. OMPs were analysed using a Voyager DEpro MALDI- TOF-MS (Applied Biosystems). Mass spectra were internally SDS-PAGE of OMPs calibrated against autologous trypsin peptide peaks. Data OMP samples were adjusted to 1.0 mg/ml in 2 sample were collected over the mass range 800–4000 Da, using a buffer [0.125 M Tris-HCl (pH 6.8), 20% (v/v) glycerol, minimum signal-to-noise ratio filter of 10. OMPs were ......................................................................................................................................................................................................................................... 3 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... identified from the peptide mass fingerprinting (PMF) output from lane 5 (PM684) and lane 8 (PM982) were correctly by the MASCOT (Matrix Science) search engine. The data- identified as OmpA and OmpH by PMF. It was noted that base search was restricted to the phylum Proteobacteria and proteins in the HMW OMP region (85–100 kDa) formed allowed for a maximum of one missed cleavage, modification three groups, comprising either three weakly expressed by carbaridomethylation, variable modification of methion- OMPs (Fig. 1, lanes 1, 2 and 5), two weakly expressed ine residues by oxidation and a positive peptide charge of 1. OMPs and one lower MW, strongly expressed OMP (Fig. 1, lanes 3, 4 and 8) or five moderately expressed Prediction of OMPs from genome sequence OMPs (Fig. 1, lanes 6 and 7). When isolates were clustered OMPs identified by MS were compared with proteins pre- by HMW OMP homology, there was an association dicted to have an OM subcellular localization from the com- between HMW OMP-type and disease-type (Fig. 2). plete genome sequence of P. multocida strain Pm70 Isolates responsible for invasive diseases (avian PM144 and (GenBank accession no. AE004439). To maximize cover- PM246, fowl cholera; porcine PM684, PAR) formed a age of the OM proteome, OMP predictions were generated single distinct cluster. The remaining isolates formed two from the combined results of analysis of the Pm70 genome clusters associated with pneumonic pasteurellosis. using PA v.3.0 (http://www.cs.ualberta.ca/~bioinfo/PA/) MALDI-TOF-MS identified that OMPs in the HMW and PSORTb v.2.0 (http://www.psort.org/psortb) software. region were mainly involved in iron acquisition. The two All PSORTb OM subcellular localization predictions had a groups of isolates associated with pneumonic disease confidence value .9.0. expressed different iron-acquisition proteins depending on the HMW OMP-type. The group comprising both bovine isolates (PM564 and PM632) and ovine isolate PM982 Results expressed a haemoglobin-binding protein (HgbA) and SDS-PAGE and MALDI-TOF-MS of OMPs from iron-rich transferrin-binding protein (TbpA). The second pneumonic culture group, comprising porcine isolate PM734 and ovine isolate PM966, expressed haem-acquisition receptors HemR and OMP profiles comprised two major proteins (OmpA and PfhR. The OMP profiles of PM734 and PM966 were OmpH) and 11–14 minor proteins (Fig. 1). Positive controls Figure 1. Coomassie blue-stained SDS-PAGE gel showing OMP profiles of Pasteurella multocida isolates of avian, bovine, porcine and ovine origin. Isolates were grown in iron-rich media. Lane numbers are given at the top of the gel. Isolate designation and host of origin is stated at the bottom. Lane 9 shows the molecular weight (MW) standards (kDa). Labelled bands correspond to OMPs of interest discussed in the main text and were identified by MALDI-TOF-MS. ......................................................................................................................................................................................................................................... 4 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... Figure 2. SDS-PAGE profiles of Pasteurella multocida isolates grown in iron-rich media, clustered by homology of HMW OMPs. Isolates associated with invasive diseases clustered into a single group. Pneumonic isolates formed two distinct clusters. Disease type is indicated at the top of the gel. Isolate designation and host of origin is stated at the bottom. MW standards (kDa) are indicated. The HMW OMP region is highlighted, as are proteins PM1069 and OMP16 that were present in all isolates but demonstrated MW variation. generally very similar, and both also expressed a protein with iron-transport OMP (Fig. 3). HemR and PfhR were common a moderate degree of homology to a Shewanella pealeana to all invasive disease isolates. Porcine isolate PM684 expressed type IV pilus biogenesis protein PilZ (Fig. 1). Only one an additional OMP, HasR, which was not identified from the HMW OMP of the invasive disease cluster was successfully invasive avian isolates, but was expressed by the other porcine identified by MS at this point and was an organic solvent tol- isolate PM734, which was associated with pneumonic disease. erance (Ost) protein. HMW OMP expression by porcine isolate PM734 increased in response to iron limitation, and six bands were SDS-PAGE and MALDI-TOF-MS of OMPs from iron-limited visible compared with five bands in iron-rich culture. culture Expressed iron-uptake OMPs identified were HasR, HemR, PfhR and PM0741 (Fig. 4, Tables 2 and 3). Ovine isolate Although invasive disease isolates PM144, PM246 and PM966, which had a very similar OMP profile to PM734 PM684 and pneumonic isolates PM734 and PM966 in iron-rich culture, had reduced HMW OMP expression formed distinct groups by HMW OMP similarity following (four bands visible), although expression of HemR, PfhR iron-rich culture, expression of HMW OMPs by these and PM0741 was confirmed (Fig. 4, Tables 2 and 3). groups was altered in response to iron-limitation (Fig. 3). Isolates that were previously similar by HMW OMP-type OMPs demonstrating MW variation had different HMW OMP profiles depending on the host of origin (Fig. 4, Tables 2 and 3). In contrast, OMP MS of proteins expressed following iron-rich or iron-limited expression by the pneumonic disease cluster comprising culture identified four proteins that were present in isolates bovine isolates PM564, PM632 and ovine isolate PM982 from different host species but in variant MW forms. A con- was unaffected by iron limitation. In particular, there was served band of 47.3 kDa was identified as hypothetical no significant change in expression of iron-acquisition pro- protein PM1069 (Fig. 1). MW variation of PM1069 was teins TbpA or HgbA. observed for isolates from different hosts and was most MS of proteins in the HMW region revealed that several notable for ovine isolate PM982 which possessed a iron-acquisition OMPs were expressed by isolates from the 44.2 kDa MW variant (Fig. 2). A 17.9 kDa conserved invasive disease group and the PM734/PM966 pneumonic protein was identified as OMP16 (Fig. 1). PM564 expressed group. These included haem-acquisition OMPs HasR, an 18.1 kDa form, and PM246 a 19.2 kDa form (Fig. 2). A HemR, PfhR and hypothetical protein PM0741, a putative 40.7 kDa OMP, HexD, was conserved across species but was ......................................................................................................................................................................................................................................... 5 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 3. Coomassie blue-stained SDS-PAGE showing OMP profiles of Pasteurella multocida isolates of avian, bovine, porcine and ovine origin, following growth in iron-limited conditions. Lane numbers are given at the top of the gel. Isolate designation and host of origin is stated at the bottom. Lane 9 shows the MW standards (kDa). Labelled bands denote OMPs of interest discussed in the main text and were identified by MALDI-TOF-MS. only weakly expressed by avian isolates and porcine isolate inorganic ion and metabolic trafficking (12 OMPs), and PM684 (Fig. 3). Expression of PM1069, OMP16 and secretion and intracellular transport (8 OMPs). A quarter HexD was not affected by iron limitation. MS of HMW of predicted OMPs were of unknown function. OMPs revealed that OMP87, which is not associated with Comparison of expressed and predicted P. multocida OM iron-uptake, was conserved across isolates from all four proteome hosts (Fig. 3). However, expression levels were difficult to compare due to the tight clustering of bands and altered In total, 22 different OMPs were shown to be expressed by expression of proteins in the HMW region under iron-rich P. multocida isolates from different hosts under iron-rich or or iron-limited conditions. iron-limited growth conditions. A small number of proteins identified by MS were not identified by the proteome predic- The in silico predicted OM proteome tion software, including the major protein OmpA and Analysis of the P. multocida strain Pm70 genome sequence OMP16. This may have been due to differences in nomencla- by PSORTb and PA software identified a total of 79 proteins ture. Prediction software identified multiple occurrences of predicted to have an OM localization. Thirty-nine OMPs OmpH (three occurrences), Hsf (two occurrences) and LspB were identified by both prediction algorithms, whereas 11 (two occurrences). Expression of all definitively identified OMPs were predicted only by PSORTb and 29 by PA iron-uptake OMPs predicted from the Pm70 genome was con- alone. Predicted OMPs represented 12 functional classes by firmed in different isolates. However, whereas only three clusters of orthologous groups (COGs) (Fig. 5). The major definitive iron-acquisition OMPs were predicted from the groups identified were cell envelope biogenesis (31 OMPs), Pm70 genome (HasR, HemR and PfhR), MS identified five ......................................................................................................................................................................................................................................... 6 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... Figure 4. OMP profiles of Pasteurella multocida isolates cultured in iron-rich or iron-limited media resolved by Coomassie blue-stained SDS-PAGE. Isolates are grouped by HMW OMP similarity under iron-rich culture conditions. Isolate ID, host species, disease type, iron availability and MW standards (kDa) are indicated. Numbered bands denote OMPs identified by MALDI-TOF-MS. Corresponding band identities are shown in Tables 2 (iron-rich culture) and 3 (iron-limited culture). iron-uptake OMPs (HasR, HemR, PfhR, HgbA and TbpA). Eight hypothetical proteins were predicted to have HgbA and TbpA were not predicted from the avian Pm70 iron-uptake functions. Expression of hypothetical OMPs genome, but were expressed by bovine isolates PM564 and PM0300, PM0336 and PM0741 (putative iron-uptake PM632 and ovine isolate PM966. OMPs by COGs classification) was confirmed (Fig. 4, ......................................................................................................................................................................................................................................... 7 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Table 2. Identification of selected Pasteurella multocida OMPs from iron-rich culture, determined by Mascot and BLASTP analysis Band Accession No. Protein name Molecular Protein Matched Coverage identity weight score peptides (%) ....................................................................................................................................................................................................................................... PM144.1 15214145 47kDa OMP precursor 46 032.7 219 19 56 15602934 PM1069 47 831.6 217 19 54 PM144.2 49182350 OmpA 37 785.4 85 17 61 PM246.1 15602651 PM0786 38 121.6 129 25 73 49182354 OmpA 38 125.6 102 22 67 PM564.1 15602165 PM0300 110 044.6 153 42 47 33340606 HgbA 110 664.9 125 37 44 PM564.2 13027667 TbpA 89 377.0 547 33 47 PM564.3 6977946 OMP 16 protein 14 207.0 180 6 73 PM632.1 15214145 47 kDa OMP precursor 46 032.7 108 18 60 15602934 PM1069 47 831.6 106 18 58 PM632.2 6977946 OMP 16 protein 14 207.0 140 8 82 PM684.1 25008882 LPS-assembly precursor (Ost) 90 819.4 88 26 40 PM684.2 15602651 PM0786 38 121.6 114 20 66 49182354 OmpA 38 125.6 85 16 54 PM684.3 2853254 OmpH 34 656.9 105 18 54 PM734.1 15602441 HemR 85085.8 330 26 39 PM734.2 157962851 PilZ [S. pealeana ATCC 92 441.9 78 28 41 700345] PM966.1 7716525 Putative TonB-dependent 110 287.5 79 33 35 receptor PM966.2 15602441 HemR 85 085.8 187 22 33 15601905 PfhR 81 510.1 182 24 30 PM966.3 15214145 47 kDa OMP precursor 46 032.7 84 20 60 15602934 PM1069 47 831.6 82 20 58 PM966.4 157962851 PilZ [S. pealeana ATCC 92 441.9 76 27 40 700345] PM982.1 110725170 TbpA 89 399.2 329 36 53 PM982.2 15214145 47 kDa OMP precursor 46 032.7 78 18 57 15602934 PM1069 47 831.6 76 18 55 PM982.3 99083546 OMP 38 821.3 123 15 40 2853254 OmpH 34 656.9 83 13 39 Band identities correspond to Fig. 4. Bands in bold are the nearest characterized protein identification when the highest scoring OMP was a poorly characterized or hypothetical protein (HP). Tables 2 and 3). A further five hypothetical proteins with between animal species. However, the role of specific adhe- probable iron receptor functions were predicted from the sins in host colonization and disease has not been well 21, 22 Pm70 genome (PM0337, PM0745, PM1081, PM1282, characterized. Study of a bovine P. multocida strain PM1428), but expression was not confirmed. implicated fibronectin (Fn) as a specific receptor for P. multocida and identified five OMPs involved in binding to host Fn. Fn is a dimer that occurs in different isoforms and has eight functional domains that may be exploited as Discussion receptors for bacterial colonization. Two classes of Fn have Candidate adhesins of P. multocida been identified—a soluble form present in body fluids and 23 20 an ECM form. Dabo et al. showed that bovine strains It has been proposed that the broad host range of preferentially bound the N-terminal Hep 1 domain. In the P. multocida is due to colonization by adherence to extra- current study, three of the OMPs identified previously, cellular matrix (ECM) components that are ubiquitous ......................................................................................................................................................................................................................................... 8 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... Table 3. Identification of selected Pasteurella multocida OMPs from iron-limited culture, determined by Mascot and BLASTP analysis Band ID Accession No. Protein name Molecular weight Protein score Matched peptides Coverage (%) ........................................................................................................................................................................................................................................ PM144.1 145632020 CGSHi22421_00657 90 430 90 13 21 [H. influenzae] PM144.2 15603857 HP PM1992 87 822 250 28 45 27527748 Omp87 87688 222 26 41 PM144.3 15601905 PfhR 81 510 179 24 39 PM144.4 15603595 PlpB 30 276 110 12 59 PM144.5 15602863 HP PM0998 30 915 89 9 44 PM246.1 15602201 HP PM0336 114 285 224 29 35 PM246.2 15603487 HasR 96 079 331 35 50 PM246.3 15603857 HP PM1992 87 822 132 20 32 27527748 Omp87 87 688 120 19 29 PM246.4 15601905 PfhR 81 510 264 29 51 PM246.5 15214145 47 kDa OMP precursor 46 033 212 21 69 15602934 HP PM1069 47 832 209 21 67 PM246.6 15602651 HP PM0786 38 122 172 21 51 49182354 OmpA 38 126 147 18 45 PM246.7 15603408 HP PM1543 26 864 97 11 60 PM246.8 6977946 OMP 16 protein 14 207 115 8 90 PM564.1 13027667 TbpA 89 377 254 28 45 PM632.1 33340606 HgbA 110 665 148 22 29 PM632.2 15603857 HP PM1992 87 822 307 33 53 27527748 Omp87 87 688 293 32 53 PM632.3 13027667 TbpA 89 377 350 36 54 PM632.4 15602643 HexD 43 034 123 14 52 PM632.5 15602863 HP PM0998 30 915 86 9 41 15214146 24 kDa OMP precursor 28 719 78 8 35 PM684.1 15603487 HasR 96 079 369 37 51 PM684.2 15602668 HP PM0803 91 050 147 22 32 PM684.3 15601905 PfhR 81 510 271 30 55 PM734.1 15603487 HasR 96 079 248 29 47 PM734.2 27527748 Omp87 87 688 119 18 32 PM734.3 15601905 PfhR 81 510 268 30 50 PM734.4 15602643 HexD 43 034 131 15 60 PM966.1 15603857 HP PM1992 87 822 302 32 50 27527748 Omp87 87 688 288 31 51 PM966.2 15602606 HP PM0741 89 716 114 19 30 PM966.3 15601905 PfhR 81 510 182 24 40 PM966.4 15602863 HP PM0998 30 915 107 9 40 15214146 24 kDa OMP precursor 28 719 98 8 34 PM982.1 110725170 TbpA 89 399 250 30 48 Band identities correspond to Fig. 4. Bands in bold are the nearest characterized protein identification when the highest scoring OMP was a poorly characterized or hypothetical protein (HP). PM1069 (P80603), OMP87 and OMP16, were conserved present across different isolates but displayed MW hetero- between isolates from different host species, providing evi- geneity. OMPs that display MW heterogeneity across isolates dence of the importance of these OMPs to the broad host from different hosts are of particular interest, as the observed range of P. multocida. OMPs PM1069 and OMP16 were variation may have arisen due to selective pressures acting on ......................................................................................................................................................................................................................................... 9 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 5. COGs classification of OMPs predicted from the OM proteome of Pasteurella multocida strain Pm70. A total of 79 OMPs were predicted using PSORTb and PA software. these OMPs within different host niches. These proteins are disease. Other authors have previously speculated that therefore useful candidates for further study as biofilm formation by P. multocida may be important in host-specificity determinants. For example, MW hetero- establishing disease, but this area has not been well geneity between these OMPs could reflect recognition of studied. Fn-types encountered in different host niches during invasive HMW OMP profile and host specificity or pneumonic disease, variation in the abundance of Fn iso- forms found within particular host species, or recognition of When isolates were cultured in iron-rich media, they clus- different Fn domains. tered into three groups by HMW OMP homology and Pilus structures expressed by P. multocida have been there was an association between HMW OMP-type and implicated as the primary mechanism for colonization of disease, as all invasive disease isolates clustered into a nasal epithelia, but there are few studies to support this single group. It is possible that homology of HMW OMPs hypothesis. A protein with homology to the type IV pilus expressed in a nutrient-rich environment reflects an epide- assembly protein PilZ of S. pealeana was present in the miological relationship between these isolates. Nutrient- OMP profile of PM734 and PM966. However, other poten- rich culture may elicit a basal state of OMP expression, as tial pilus components, such as those comprising the OM inte- there is no pressure for increased expression of a particular gral O-ring, were not identified. In addition, putative class of uptake system. This would explain why isolates filamentous adhesin Hsf and haemagglutinins PfhB1 and from different host species had similar HMW OMP profiles PfhB2 were predicted from the Pm70 genome and may be in a nutrient-rich environment, but had dissimilar HMW important virulence determinants, but were not detected in OMP profiles under iron-limitation stress. the present study. This could have been due to incomplete The results also suggested that expression or regulation of identification of the OM proteome, lack of expression in iron-uptake OMPs may be adapted for survival within par- vitro or because the vigorous methods involved in sarcosyl ticular host species or specific host niches. Avian isolates extraction are inappropriate for the isolation of fragile fila- PM144 and PM246 were both associated with septicaemia mentous surface structures. and had very similar OMP profiles under iron-limited con- Expression of type IV pili by P. multocida has been ditions. However, porcine isolates PM684 and PM734 characterized for serogroups A, B and D, and is responsible were associated with diseases (PAR and pneumonia respect- for twitching motility. Twitching motility is an important ively) that occur at different sites within the host, and had step in biofilm formation, for example during lung infections different HMW OMP profiles under iron limitation. by Pseudomonas aeruginosa in cystic fibrosis patients. It is Haem is the most abundant iron source in birds and feasible that biofilm formation is also important during lung mammals. Avian isolates PM144, PM246, porcine isolates infections by P. multocida, which would link expression of PM684, PM734 and ovine isolate PM966 expressed an array type IV pili to the ability of isolates to cause pneumonic of OMPs involved in the acquisition of iron via haem-uptake ......................................................................................................................................................................................................................................... 10 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... (HasR, HemR and PfhR). HasR is a haemophore receptor. proteome was expressed. While the expressed OM proteome Haemophore systems typically involve type I secretion of is expected to differ from the complete OM proteome due to the haemophore HasA into the extracellular environment, environmental and temporal regulation of expression, it has binding of host haem and delivery to the transmembrane been suggested that the majority of OMPs could fail detec- channel HasR. However, less efficient haem-uptake via tion due to low levels of expression. OMPs may also be direct binding of haem by HasR may also occur. BLAST mistakenly predicted from genes with unusual localization analysis revealed that the Pm70 genome does not contain signal sequences or from pseudogenes. In this study, three the hasA gene suggesting that at least some P. multocida iso- OmpH proteins (OmpH_1, OmpH_2, OmpH_3) were pre- lates utilize a HasA-independent mechanism. HemR and dicted. However, comparison against the OmpH_1 amino PfhR are thought to be haem-transport porins which bind acid sequence by BLAST analysis revealed that OmpH_2 different haem-containing compounds directly. The presence and OmpH_3 contain sequence gaps and are potentially of multiple haem-uptake mechanisms may broaden the range non-functional pseudogenes. of haem sources accessible by these isolates. In conclusion, 22 OMPs were identified from the OM pro- Haem-uptake systems are often associated with haemoly- teome of eight P. multocida isolates recovered from different sins that liberate haem from host erythrocytes and other cell host species. Expression of several hypothetical OMPs pre- types. P. multocida is described as non-haemolytic under dicted from the P. multocida Pm70 proteome was confirmed aerobic conditions. However, recent studies have reported by PMF. A variety of potential host colonization factors were production of an inner membrane-bound haemolysin identified, some of which were present in isolates from differ- AhpA, expressed under anaerobic conditions in vitro ent host species and occurred as MW variants (e.g. OMP16, 30, 31 and is most active against avian red blood cells. PM1069). HMW OMPs were mainly involved in iron Furthermore, a haemolysin secretion and activation protein acquisition. In iron-replete conditions, isolates clustered LspB was predicted from the Pm70 genome by both into three groups by HMW OMP profile, and there was an PSORTb and PA. The role of the putative AhpA virulence association between these groups and disease. However factor in host pathogenesis is unknown, but it is conceivable within these clusters, isolates from different host species that haemolysin production could enhance haem acquisition regulated iron-acquisition OMPs differently in response to under certain in vivo conditions and could be associated with iron limitation, suggesting that similar isolates may have increased virulence of strains. evolved alternate mechanisms for regulating expression of Bovine isolates PM564, PM632 and ovine isolate PM982 iron-acquisition OMPs. This may reflect adaptation for sur- expressed a unique set of iron-acquisition OMPs, HgbA and vival within different host niches. TbpA, which were not expressed by avian or porcine isolates and were not predicted from the avian Pm70 genome. Acknowledgements Acquisition of iron from host transferrin (Tf) typically involves two components, a lipoprotein TbpB, which is a I wish to thank the staff at the Glasgow Biomedical Research high-specificity receptor for host Tf, and a 100 kDa tonB- Centre and the Sir Henry Wellcome Functional Genomics dependent iron transport channel TbpA. TbpB-independent Facility for their support during this project, with special Tf binding can occur, but is less efficient. The present thanks to Dr. Robert L. Davies, Dr. Richard Burchmore study concurs with previous reports that P. multocida and Jay Jayawarzdena. expresses an 82 kDa form of TbpA and does not express TbpB at all, and furthermore, that TbpA is expressed only by bovine and ovine isolates. In this study, TbpA was con- Funding stitutively expressed in iron-rich and iron-limited conditions. Funding for this project was provided by the Faculty of This may be because the P. multocida tbp locus lacks the Biomedical and Life Sciences, University of Glasgow. Fur-regulated promoter which is usually upstream of tbpB and is therefore not regulated by iron availability. The presence of a neighbouring insertion sequence suggests that References the tbpA gene may have been acquired by horizontal 1. Rimler RB, Rhoades KR (1989) Pasteurella multocida.In CF Adlam, JM Rutter, transmission. eds, Pasteurella and Pasteurellosis. London: Academic Press, pp. 37–73. 2. Chanter N, Rutter JM (1989) Pasteurellosis in pigs and the determinants of Comparison of in vitro and in silico OM proteomics virulence of toxigenic Pasteurella multocida. In CF Adlam, JM Rutter, eds, The OMPs identified in this study represented around one- Pasteurella and Pasteurellosis. London: Academic Press, pp. 161–195. quarter of the OMPs predicted from the Pm70 genome. 3. Frank GH (1989) Pasteurellosis of cattle. 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Anal Biochem 87: 206–210. J Proteome Res 2: 303–311. ........................................................................................................................................................................................................................................ Submitted on 30 September 2008; accepted on 19 January 2009; advance access publication 17 February 2009 .........................................................................................................................................................................................................................................

Journal

Bioscience Horizons – Oxford University Press

Published: Mar 17, 2009

Keywords: Key words Pasteurella multocida outer membrane protein host specificity iron acquisition

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